Cross Talk between tRNA and rRNA Synthesis in Saccharomyces cerevisiae

Abstract

Temperature-sensitive RNA polymerase III (rpc160-112and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [³³P]orthophosphate and coextracted with [³H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25°C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37°C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: the rpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25°C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

ACKNOWLEDGMENTS

We thank Jean Labarre and Jean-Marie Buhler for useful suggestions, Michel Werner and anonymous reviewers for improving the manuscript, and André Sentenac for his kind support.

J.-F.B. had a fellowship from the Fondation de la Recherche Médicale, F.N. held a European Marie Curie Fellowship, and O.G. was supported by the Institut de Formation Supérieure Biomédicale. This work was partly funded by the European Training and Mobility Program (grant FMRX-CT96-0064).